Enzymes and micro organisms with amidase activity which hydrolyze polyamides

ABSTRACT

The present invention relates to a process for the enzymatic hydrolysis of polyamides 6.6 to give adipic acid monomers and hexamethylenediamine monomers. The present invention further relates to an enzyme with amidase activity particularly towards substrates of the oligomer type derived from PA 6.6 and/or PA 6, said enzyme being characterized in that it consists of a peptide sequence corresponding to SEQ ID NO: 1 in the attached sequence listing and/or at least one polypeptide homologous to this sequence. The invention further relates to the DNA coding for said enzyme and to the biological precursors thereof The invention further relates to the microorganisms capable of producing this enzyme and to the hydrolysis process in which this enzyme and/or these microorganisms are applied.

TECHNICAL FIELD

The present invention relates in general terms to the enzymatichydrolysis of amides, especially secondary amides.

More precisely, the invention relates first of all to a process for theenzymatic hydrolysis of substrates of the polyamide 6.6 type to give thetwo comonomers, A and B, of said substrates. The invention furtherrelates to enzymes and/or microorganisms which are capable of being usedin the enzymatic hydrolysis of amide groups, preferably on substratescontaining at least one amide group, for example polyamides (PA). Theinvention further relates to the genetic tools expressing these enzymes.

PRIOR ART

In this field, SMITH R. et al. are the authors of an article publishedin “Journal of Biomedical Materials Research (1987), vol. 21, p.991-1003” and disclose the bringing of samples of polyamide 66 labeledwith carbon 14 into contact with enzymes of the papain, trypsin andα-chymotrypsin type. These known polypeptides degrade polyamide 66slightly, but the hydrolysis is not sufficiently significant to be ableto be exploited on the industrial scale.

It is also known, through the article by KINOSHITA et al. (Eur. J.Biochem. 116, 547-551, 1981), that Flavobacterium sp KI 72 is able toproduce a first enzyme (E₁) which catalyzes the hydrolysis of cyclicdimers of 6-aminohexanoic acid to linear dimers of this same acid, and asecond enzyme (E₂) which is capable of converting this linear dimer totwo molecules of 6-aminohexanoic acid or aminocaproic acid. Theenzymatic pathway in question is summarized below.

The activity of the linear amidase E₂ is optimal for the dimers anddecreases as the degree of polymerization increases, no longer beingsignificant beyond oligomers with a degree of polymerization (DP_(n)) of7.

An enzyme E₃ active towards cyclic and linear oligomers of6-aminohexanoic acid of DP_(n)≧3 (PA 6) is also known. E₃ is describedby NEGORO et al. in “Journal of Bacteriology, Dec. 1992, vol. 174, no.24, p. 7948-7953”. E₃ also originates from Flavobacterium sp KI 72.

In their article published in “Journal of Bacteriology, June 1989, p.3187-3191, vol. 171 no. 6”, TSUCHIYA et al. teach that a high degree ofhomology exists between the enzymes E₁ from Flavobacterium sp KI 72 andone of the enzymes derived from Pseudomonas sp NK 87. These enzymes E₁and homologs, and the enzymes E₂, more particularly the latter, are saidto be active on oligomers or polyamides (PA 6) of the formula

—CONH—(CH₂)₅—CO_(n)NH—

where: 2≦n≦20.

The disadvantage of these enzymes derived from Flavobacterium orPseudomonas is that they have relatively low specific activities towardsoligomers, said activities amounting to at most only 1.05 micromol ofaminocaproic acid produced per minute and per milligram of protein froma substrate consisting of a trimer. Furthermore, these enzymes arespecific for homo-oligomers.

It is thus apparent that the prior art does not comprise means for theenzymatic hydrolysis of amide groups which have a high performance, areviable and can be applied to amides, including especially secondaryamides, of a variety of types, particularly of the co-oligomer and/orhomo-oligomer type.

DISCLOSURE OF THE INVENTION

Therefore, after lengthy and laborious research, the Applicant succeededin isolating and characterizing a novel enzyme of the amidase typeformed by one or more polypeptides which, in particular, can be derivedfrom novel microorganisms isolated from the biotype and/or novelrecombinant microorganisms obtained from these natural microorganisms.

Consequently, the present invention relates firstly to an enzymatichydrolysis process and secondly to an enzyme.

The process according to the invention for the hydrolysis of(poly)amides characterized in that:

enzymatic hydrolysis is carried out on substrates comprising(poly)amides of the following formula (I):

in which:

A and B are monomer units,

R¹ and R³ are identical or different—preferably different—divalentradicals representing a substituted or unsubstituted, linear or branched(cyclo)alkylene, an arylene or an arylalkylene, the aromatic radicalsoptionally being polycondensates and the number of carbons in thealkylenes being greater than or equal to 4, preferably between 4 and 12,

R² corresponds to identical or different—preferably identical—radicalsselected from hydrogen and/or alkyl radicals advantageously having from1 to 6 carbons,

X is:

either X¹=OH, OM or OR⁴, where M is selected from metals, preferablyalkali metals and alkaline earth metals, and R⁴ is a linear or branchedalkyl containing from 1 to 6 carbon atoms,

or X²=

where R² and R³ are as defined above and R⁵ and R⁶, which are identicalor different, have the same definition as that given above for R²,

Y is:

either Y¹=hydrogen,

or Y²=

where R¹ is as defined above and Z is hydrogen, M¹ defined in the sameway as M, or R⁴,

with the following conditions:

-a- if X=X¹,then Y=Y¹or Y²,

-b- if X=X², then Y=Y²,

and, finally, p is between 1 and 4, preferably between 1 and 3; and

it is capable of converting the above-mentioned substrates (I) tomonomers A, on the one hand, and monomers B, on the other.

The present invention further relates to a family of enzymes of theamidase type which are capable (inter alia) of being used in the processdefined above.

The enzyme belonging to the family according to the invention is anamidase characterized in that:

-{circle around (5)} →it is active, especially towards substrates of the(poly)amide type having the following formula:

in which:

A and B are monomer units,

R¹ and R³ are identical or different—preferably different—divalentradicals representing a substituted or unsubstituted, linear or branched(cyclo)alkylene, an arylene or an arylalkylene, the aromatic radicalsoptionally being polycondensates and the number of carbons in thealkylenes being greater than or equal to 4, preferably between 4 and 12,

R² corresponds to identical or different—preferably identical—radicalsselected from hydrogen and/or alkyl radicals advantageously having from1 to 6 carbons,

X is:

either X¹=OH, OM or OR⁴, where M is selected from metals, preferablyalkali metals and alkaline earth metals, and R⁴ is a linear or branchedalkyl containing from 1 to 6 carbon atoms,

or X²=

where R² and R³ are as defined above and R⁵ and R⁶, which are identicalor different, have the same definition as that given above for R²,

Y is:

either Y¹=hydrogen,

or Y²=

where R¹ is as defined above and Z is hydrogen, M¹ defined in the sameway as M, or R⁴, with the following conditions:

-a- if X=X¹, then Y=Y¹or Y²,

-b- if X=X², then Y=Y²,

and, finally, p is between 1 and 4, preferably between 1 and 3;

-{circle around (2)} →and it is capable of converting theabove-mentioned substrates (I) to monomers A, on the one hand, andmonomers B, on the other.

Among this family of enzymes according to the invention, it isadvantageous to isolate an enzyme with amidase activity which comprisesthe peptide sequence as shown in the attached sequence SEQ ID NO : 1, ora homologous peptide sequence having a homology of at least 80%,preferably at least 90% and particularly preferably at least 95% withSEQ ID NO: 1.

According to one advantageous characteristic of the invention, thisenzyme has amidase activity particularly towards substrates of the(poly)amide type having the following formula (II):

in which:

R²and R³ are as defined above,

U and V respectively have the same definitions as those given above forX¹ and Y¹ in formula (1) given in claim 1,

and q=1 to 8.

The invention initially arose from the isolation of a wild-type strainwhich produced the enzyme, namely: Comamonas acidovorans N 12.

The identification of this wild-type strain is the result of along-winded screening operation. This identification was effected on thebasis of the morphological, cultural, biochemical and antigenicproperties which could be determined by reference to the officialinternational criteria of microbiological taxonomy.

The Applicant's merit is not limited to the isolation of this wild-typestrain, but also extends to the isolation of the amidase defined above.The microorganism Comamonas acidovorans N 12 is not the exclusivebiological precursor of this amidase. In fact, it is also necessary toconsider all recombinant microorganisms and wild-type strains which havea similar enzymatic activity.

The recombinant microorganisms are those which possess in their genome aDNA sequence coding for the amidase considered in the present invention.

According to another of its aspects, the invention further relates to anenzymatic hydrolysis process in which the above-mentioned enzyme and/orthe above-mentioned wild-type microorganism and/or at least one of itsabove-mentioned recombinant microorganisms are used.

According to the invention, the enzyme substrates and/or theirbiological precursors (microorganisms/recombinant microorganisms) arepolyamides and, more precisely, oligomers whose repeat polymerizationgroup is a secondary amide group —CO—NH—.

These oligomers have repeat units of formulae (I) and/or (II). Therepeat units (I) are advantageously formed of two monomer units, A andB, which are respectively dicarbonyl and diamine units and are joinedtogether by a secondary amine group.

In one preferred modality of the invention:

the monomer A is a residue

where r is between 4 and 12 and is preferably equal to 4,

and the monomer B is a residue

—HNCH₂_(s)NH—

where s is between 4 and 12 and is preferably equal to 6.

Thus, for the substrates (I), a preferred example of a dimer is thatformed by

The corresponding polyamide is PA 6.6.

The repeat units (II), on the other hand, are preferably formed bymonomer units denoted as {circle around (a)}—{circle around (c)}—{circlearound (b)}, in which c is a skeleton of the amino acid type whose endgroups {circle around (a)} and {circle around (b)} are carboxyl andamino groups respectively.

A typical example of {circle around (a)}—{circle around (c)}—{circlearound (b)} is a derivative of c-aminocaproic acid, the monomer ofpolyamide 6 (PA 6).

The following may be mentioned among the oligomers of the polyamide typewhich are suitable for the invention:

the polyamide oligomers obtained by the polycondensation of saturatedaliphatic carboxylic diacids having from 6 to 12 carbon atoms withsaturated aliphatic primary diamines having from 6 to 12 carbon atoms,

the polyamino acid oligomers obtained either by the directhomopolycondensation of an ω-aminoalkanoic acid containing a hydrocarbonchain having from 4 to 12 carbon atoms, or by the hydrolytic opening andpolymerization of the lactams derived from these acids,

the copolyamide oligomers obtained from the starting monomers of theabove-mentioned polyamides, it also being possible for the acidcomponent of these copolyamides to consist partly of an aromatic acidsuch as terephthalic acid and/or isophthalic acid,

and mixtures of these polyamide oligomers.

The following may be mentioned as illustrative examples of thepolyamides obtained by the polycondensation of diacids and diamines:

polyamide 4,6 (polymer of tetramethylenediamine and adipic acid),

polyamide 6,6 (polymer of hexamethylenediamine and adipic acid) (PA6.6),

polyamide 6,9 (polymer of hexamethylenediamine and azelaic acid),

polyamide 6,10 (polymer of hexamethylenediamine and sebacic acid),

polyamide 6,12 (polymer of hexamethylenediamine and dodecanedioic acid).

The following may be mentioned as illustrations of suitable polyaminoacids:

polyamide 4 (polymer of 4-aminobutanoic acid or γ-butyrolactam),

polyamide 5 (polymer of 5-aminopentanoic acid or δ-amylolactam),

polyamide 6 (polymer of ε-caprolactam),

polyamide 7 (polymer of 7-aminoheptanoic acid),

polyamide 8 (polymer of capryllactam),

polyamide 9 (polymer of 9-aminononanoic acid),

polyamide 10 (polymer of 10-aminodecanoic acid),

polyamide 11 (polymer of 11-aminoundecanoic acid),

polyamide 12 (polymer of 12-aminododecanoic acid or laurolactam).

The following may be mentioned as illustrative examples of copolyamides:

polyamide 6,6/6,10 (copolymer of hexamethylenediamine, adipic acid andsebacic acid),

polyamide 6,6/6 (copolymer of hexamethylenediamine, adipic acid andcaprolactam).

Without implying a limitation, the preferred substrates according to theinvention are polyamide oligomers obtained by the polycondensation ofdiacids A and diamines B, particularly oligomers of PA 6.6.

The number of monomers A and B, on the one hand, or {circle around(a)}—{circle around (c)}—{circle around (b)}, on the other, in thesubstrates (I) and (II) according to the invention is advantageouslybetween 2 and 8, preferably between 2 and 6.

It should be pointed out that this number of monomers A and B in theoligomer or polymer molecules will also be called DP_(n) in the presentdisclosure.

Characteristic substrates (I) are e.g. advantageously water-solubleoligomers such as AB, ABA and ABAB.

It should be pointed out that this number of monomers A and B in theoligomer or polymer molecules will also be called DP_(n) in the presentdisclosure.

These particular substrates are among the substrates (I) and (II) whichcan be hydrolyzed by the enzyme of the invention and/or its biologicalprecursors because they possess at least one carboxyl end group.

When the enzymatic substrates are oligomers containing monomers A and B,the final hydrolysis products can be monomers A and B.

When the substrates are oligomers (II) (monomers ({circle around(a)}—{circle around (c)}—{circle around (b)}), the final hydrolysisproducts can be monomers {circle around (a)}—{circle around (c)}—{circlearound (b)}.

The enzyme according to the invention can also be characterized throughits activity and/or its affinity towards some of the above-mentionedsubstrates.

Thus, according to a first advantageous characteristic, the amidase inquestion is:

firstly active towards a substrate (I) formed by a tetramer of A and B(DP_(n)=4, i.e. p=2, X=X¹ and Y=Y¹),

and secondly capable of converting this substrate on the one hand to anoligomer of DP_(n)=3 and on the other hand to a monomer A with aspecific enzymatic activity (U_(s))—expressed in μmol of hydrolyzedABAB×h⁻¹×mg⁻¹ of protein and measured under given conditions—which isgreater than or equal to 1000.

A second advantageous characteristic of this amidase is that it isactive towards a substrate (I) formed by a trimer ABA, and capable ofconverting this trimer to a monomer A and a dimer AB with a specificenzymatic activity greater than or equal to 1000 μmol of hydrolyzedsubstrate×h⁻¹×mg⁻¹ of protein.

A third advantageous characteristic of this amidase is that it is activetowards a substrate (I) formed by a dimer of the type AB, and capable ofconverting this dimer to two monomers A and B with a specific enzymaticactivity greater than 1500 μmol of hydrolyzed substrate×h⁻¹×mg⁻¹ ofprotein.

The enzymatic activity of the pure amidase is measured under thefollowing conditions: phosphate buffer, pH 6 to 8 and temperature=30° C.

The amidase enzyme of the invention has at least one of these threenon-limiting characteristics.

As already indicated above, the use of the enzyme of the invention inthe enzymatic hydrolysis of amides can consist in using only the enzymeper se instead of the biological precursors (wild-type or recombinantmicroorganisms) which produce said enzyme, or a mixture of both.

On the subject of enzyme producers, it should be emphasized that theenzyme forming the subject of the invention is also characterized inthat it is produced by microorganisms of the type Comamonas acidovorans(N 12), preferably of the same type as the strain referenced anddeposited in the Collection Nationale de Cultures deMicro-organismes—Institut Pasteur PARIS—under no. I 1522 on Jan. 4,1995, and/or by the recombinant microorganisms as described above.

The present invention further relates to novel microorganisms capable ofproducing the amidase enzyme according to the invention, as definedabove. By virtue of this ability, the particular characteristics ofthese microorganisms are, on the one hand, a capacity to hydrolyze theamide groups of a polyamide compound comprising at least one amidegroup, and particularly oligomers. More precisely, these microorganismshave a specific selectivity and hydrolytic activity towards thesubstrates (I) and (II) defined above. In the case of the substrates(I), the oligomers are for example the water-soluble oligomers mentionedabove: ABAB and/or ABA and/or AB, inter alia.

More precisely, these microorganisms can preferably consist of Comamonasacidovorans mentioned above, preferably the strain referenced anddeposited in the Collection Nationale de Cultures de Micro-organismesunder no. I 1522 on Jan. 4, 1995, or recombinant microorganisms thereof,as described above.

Advantageously, the microorganisms according to the invention arecapable of hydrolyzing at least one substrate formed by a polyamideoligomer and, more particularly, by a dimer AB, which is destined to beconverted to two monomers A and B, the microorganisms being capable ofperforming the hydrolysis.

Apart from the biological precursors of the amidase in question, thepresent further relates to the genetic material under whose control itcan be synthesized via recombinant or non-recombinant microorganisms.Consequently, the present invention relates to a DNA sequence coding foran enzyme with amidase activity, characterized in that it is selectedfrom the following sequences:

Δ the DNA sequence as represented by SEQ ID NO: 2 in the attachedsequence listing and coding for at least one enzyme with amidaseactivity,

Δ an analog of this sequence which results from the degeneracy of thegenetic code, and

Δ a DNA sequence hybridizing with the above-mentioned sequence or withat least one fragment thereof and coding for an enzyme with amidaseactivity.

The enzymes resulting from the expression of the above-mentioned DNAsequence are also included in the field of the invention.

The wild-type microorganisms (e.g. I 1522) and the recombinantmicroorganisms isolated by the Applicant each contains at least oneexpression cassette comprising the DNA sequence SEQ ID NO: 2 referred toabove, and optionally, upstream thereof, at least one promoter sequenceand at least one ribosome binding site.

The present invention further relates to a process for the hydrolysis ofsubstrates which are at least partly formed by the substrates (I) and/or(II) as defined above, characterized in that it consists in using atleast one enzyme and/or at least one of the microorganisms as presentedabove.

It may be advantageous, according to the invention, to have severalenzymes with complementary spectra. Therefore, one of the variants ofthe above-mentioned process can be to have the use of at least one othertype of enzyme and/or at least one of its wild-type and/or recombinantbiological precursors. In this variant, the substrates consist at leastpartly of oligomers whose DP_(n) is less than 40, preferably 20 andparticularly preferably 12, and which are derived from polyamides atleast originating from polycondensation between diacid monomers (A) anddiamine monomers (B).

The hydrolysis process according to this variant is characterized inthat:

it produces oligomers with a degree of polymerization (DP_(n)) less thanor equal to 3 and preferably produces monomers A and B,

and the following are used:

at least one enzyme and/or at least one microorganism as defined above,

and at least one other type of enzyme and/or to at least one of itswild-type and/or recombinant biological precursors, said enzyme being:

an enzyme E₃ produced under the control of the nyl-c gene ofFlavobacterium sp KI 72,

and/or an enzyme called PAM I as defined by the attached peptidesequence SEQ ID NO : 3, it being possible, inter alia, for such anenzyme to be produced by the microorganism referenced and deposited inthe Collection Nationale de Cultures de Micro-organismes—InstitutPasteur PARIS—under no. I 1495 on Nov. 29, 1994.

Oligomers hydrolyzable by the above enzymes can be obtained by thermaland/or chemical (acid) lysis of the groups of the amide type.

Other advantageous modalities of the process according to the inventionuse especially biological precursors of the enzyme (or enzymes) and aculture medium comprising, for example:

a carbon source preferably comprising at least one compound containingat least one amide group, said carbon source optionally comprising acomplement advantageously selected from carbohydrates, sucrose beingparticularly preferred,

and optionally a compound capable of inducing enzyme production withoutbeing consumed by the biological precursors, said compound preferablybeing selected from amides.

The process according to the invention for the enzymatic hydrolysis ofamides can have numerous applications, for example in organic synthesisfor the manufacture of compounds from amide compounds or for thetreatment of materials containing polyamide.

In particular, it could be of value within the framework of regeneratingthe starting materials of polyamide polymers.

The Examples which follow provide an illustration of thecharacteristics, variants and advantages of the present inventionwithout however limiting its scope

DESCRIPTION OF THE ATTACHED FIGURES

The sequence listing is established according to WIPO standard ST 23 andcomprises:

SEQ ID NO: 1: amino acid sequence corresponding to the enzyme PAM IIaccording to the invention,

SEQ ID NO 2: DNA (pam II) coding for the amidase PAM II,

SEQ ID NO: 3: amino acid sequence corresponding to PAM I,

SEQ ID NO: 4: DNA coding for the enzyme PAM I.

FIG. 1 shows the restriction map of plasmid pXL2297 containing the gene(pam I) coding for the amidase PAM I.

FIG. 2 shows the restriction map of plasmid pXL2564 containing the gene(pam I) coding for PAM I.

EXAMPLES EXAMPLE 1: ISOLATION AND IDENTIFICATION OF THE WILD-TYPE STRAINCOMAMONAS ACIDOVORANS N 12

1.1 MICROBIOLOGICAL SCREENING

A vast microbiological screening operation enabled the strain Comamonasacidovorans N 12 to be selected from various biotopes.

1.2 SELECTION-IDENTIFICATION OF THIS MICROORGANISM:

This microorganism was selected with the aid of an amidase activity teston synthetic oligomers of PA 6.6. The evaluation of activity will begiven in detail below.

The selected microorganism was then identified on the basis of itsmorphological, physiological, biochemical and possibly antigenicproperties, in completely traditional manner, by the BacteriologyLaboratory of the INSTITUT PASTEUR.

On the basis of these results and the hydrolytic activities on a mixtureof oligomers AB, ABA, BAB and ABAB, the natural strain selected was thensubjected to a more thorough characterization.

EXAMPLE 2: THOROUGH CHARACTERIZATION OF COMAMONAS ACIDOVORANS N 12

2.1 OPTIMIZATION OF THE CULTURE CONDITIONS:

The optimum culture medium is M9YE3 having the following composition:

KH₂PO₄ 0.75 g/l K₂HPO₄.3H₂O 1.00 g/l Na₂HPO₄.12H₂O 1.00 g/l (NH₄)₂SO₄2.50 g/l Yeast extract 3.00 g/l MgSO₄.7H₂O 1.00 g/l FeSO₄.7H₂O 2.30 mg/lMnSO₄.7H₂O 2.70 mg/l CoCl₂.2H₂O 2.30 mg/l CaCl₂.2H₂O 1.50 mg/lCuSO₄.5H₂O 0.25 mg/l pH 7.2

and supplemented with 10 g/l of adipic acid salt.

The cultures are prepared in Erlenmeyer flasks filled to ⅕ of theirtotal volume. The media are incubated at 30° C. and stirred at 150 rpm.

The preculture is composed of 10 ml of LB medium:

Tryptone 10 g/l Yeast extract  5 g/l NaCl 10 g/l

inoculated with a colony originating from a Petri dish containing M9YE3agar medium and 5 g/l of a compound containing an amide group, such aspentameric to decameric oligomers of PA 6.6.

The culture is then inoculated at 1% with the optimum medium (v/v). Whenculture is complete, the cell production is determined via a dry extractobtained by the drying, overnight at 105° C., of a cell residue obtainedafter centrifugation (10 min at 12,000 g)

To obtain maximum efficacy in the hydrolysis, a compound containing atleast one amide group is advantageously introduced into the medium.

According to another preferred characteristic of the invention, thecarbon source contains, together with the soluble components, acomplement advantageously selected from carbohydrates, sucrose beingparticularly preferred.

To obtain maximum efficacy in the hydrolysis, a compound containing atleast one amide group is advantageously introduced into the medium.

2.2 ENZYMATIC HYDROLYSIS—MEASUREMENT OF ACTIVITY OF THE WILD-TYPEMICROORGANISM:

The activity is measured at 28° C. in a 10.5 mmol/l phosphate buffer atpH 7.5 and with a final volume of 1 ml.

TABLE 1 below gives the results obtained.

TABLE 1 SUBSTRATE (concentration in the test in g/l) Comamonasacidovorans N 12 (1.4) AB → A + B 280 mg of A/h.g of dry cells (2.6) ABA→ AB + A 215 mg of AB/h.g of dry cells (1.4) ABAB → BAB + A 410 mg ofBAB/h.g of dry cells

EXAMPLE 3: PURIFICATION OF THE POLYAMIDE HYDROLASE OF COMAMONASACIDOVORANS N 12 ACCORDING TO THE INVENTION

Standard protocol:

The activity of this enzyme (PAM II) is monitored by determining theactivity in the hydrolysis of ABAB to BAB+A.

3.1 GLOSSARY:

DTE = 1,4-dithioerythritol EDTA acid = ethylenediaminetetraacetic CHAPS= 3-[(3-cholamidopropyl)dimethylammonio]- propane-1-sulfonate SDS =sodium dodecylsulfate

3.2 PREPARATION OF THE ENZYMATIC EXTRACTS:

The starting material consists of cells originating from a 20-hourculture in a 2.5 I flask filled with 500 ml of M9YE3 medium described inExample 2, supplemented with 10 g/l of adipic acid salt.

FIRST EXTRACTION BY ULTRASONIC TREATMENT:

25 g of Comamonas acidovorans cells are resuspended in 75 ml of 100 mMTris-HCl buffer, pH 7.5, containing 1 mM DTE, 5 mM EDTA, 100 mM KCl and15% v/v of glycerol.

The suspension is then subjected to a discontinuous ultrasonic treatment(10% treatment, 90% rest) in melting ice for 70 min.

The suspension is then centrifuged for 1 h 30 min at 50,000 g (Beckmanultracentrifuge).

This finally gives:

→ on the one hand 90 ml of supernatant (S1) containing 21 mg/ml ofproteins, i.e. 1890 mg of proteins of specific activity 6 μmol/h/mg(11,340 units in total); and

→ on the other hand a residue (C1), which is resuspended in 30 ml of 25mM Tris-HCl buffer, pH 8, containing 2 mM DTE, 8 mM CHAPS and 15% v/v ofglycerol.

It is this residue, also containing polyamidase II activity, which issubsequently treated. It is pointed out that it is possible, after theremoval of salt from the 90 ml of supernatant in the same buffer withoutglycerol, and renewed ultracentrifugation, to purify and reconcentrateinto a new, very fine residue the polyamidase II activity which isapparently soluble in the first centrifugation. In fact, reducing thedensity of the buffer (by removing the glycerol) accelerates theprecipitation of the very fine particles. This information is consistentwith the fact that the first supernatant cannot be isolated bychromatography, for example on a MonoQ column, since the activity isfound throughout the gradient.

COMPLEMENTARY TREATMENT OF THE RESIDUE FROM THE FIRST CENTRIFUGATION:

The resuspended residue (C1) is centrifuged for 30 min at 4000 g inorder to precipitate the cells not lyzed by the first ultrasonictreatment.

The supernatant (S2) is then collected and subjected to a discontinuousultrasonic treatment (10% treatment, 90% rest) in melting ice for 20min. It is then centrifuged again for 2 h at 50,000 g and thesupernatant (S3) is collected.

PURIFICATION STEPS BY CHROMATOGRAPHY:

1st step: The supernatant S3 is chromatographed in three portions on aMonoQ HR 10/10 column (Pharmacia) equilibrated in 25 mM Tris-HCl buffer,pH 8, containing 2 mM DTE, 8 mM CHAPS and 15% v/v of glycerol. Elutionis carried out with a linear gradient from 0 to 0.6M NaCl over 60 min ata rate of 3 ml/min. The polyamidase II activity is eluted at about 0.2MNaCl as a fairly broad peak. The active fractions are pooled andconcentrated to 5 ml on a Centriprep 10 (Amicon).

2nd step: 5 ml of 25 mM Tris-HCl buffer, pH 8, containing 1 mM DTE, 8 mMCHAPS, 1.5M ammonium sulfate and 15% v/v of glycerol, are added to the 5ml collected above. These 10 ml are then chromatographed in two portionson a Phenylsuperose HR 10/10 column (Pharmacia) equilibrated in 25 mMTris-HCl buffer, pH 8, containing 2 mM DTE, 8 mM CHAPS, 1 M ammoniumsulfate and 15% v/v of glycerol. Elution is carried out with a lineargradient from 1 to 0M ammonium sulfate over 70 min at a rate of 1.25ml/min. The polyamidase activity is eluted at about 0.8M ammoniumsulfate. The active fractions are pooled and concentrated to 400 μl on aCentriprep 10 (Amicon).

3rd step: The above 400 μl are chromatographed in two portions on a TSKG3000 SW column (Supelco) equilibrated in 100 mM Tris-HCl buffer, pH7.5, containing 2 mM DTE, 8 mM CHAPS, 150 mM NaCl and 15% v/v ofglycerol, and eluted at 0.5 ml/min. The activity is found from exclusionup to a molecular weight of about 30 kDa. The most active fractions arepooled and made up to a volume of 5 ml and the salt is removed on PD10Sephadex G25 columns (Pharmacia) (2.5 ml per column) equilibrated andeluted in 25 mM Tris-HCl buffer, pH 8, containing 2 mM DTE, 8 MM CHAPSand 15% v/v of glycerol.

4th step: The 7 ml resulting from the removal of salt on PD10 arechromatographed on a MonoQ HR 5/5 column (Pharmacia) equilibrated in 25mM Tris-HCl buffer, pH 8, containing 2 mM DTE, 8 mM CHAPS and 15% v/v ofglycerol. Elution is carried out with a linear gradient from 0 to 0.6MNaCl over 30 min at a rate of 1 ml/min. The polyamidase activity iseluted at about 0.15M NaCl as a fairly broad peak. The active fractionsare pooled and, after the addition of 0.05% of SDS, are concentrated to200 μl on a Centriprep 10 and then a Centricon 10 (Amicon).

5th step: The 200 μl collected are injected onto a TSK G3000 SW columnequilibrated in 40 mM sodium sulfate, 20 mM sodium phosphate buffer, pH6.8, containing 0.25% w/v of SDS, and eluted at a rate of 0.25 ml/min.The polyamidase emerges as a narrow peak, which is monitored byelectrophoresis. This material is used for sequencing.

DETERMINATION OF THE MOLECULAR WEIGHT BY ELECTROPHORESIS:

The molecular weight, determined by polyacrylamide gel electrophoresis,varies according to the method of preparing the polyamidase II sample.

In the presence of 2.5% of SDS and 5% of mercaptoethanol, a band isfound between 36 and 40 kDa but, if heated for 5 min at 90° C., the samesample now only migrates to a molecular weight of about 150 kDa.According to its purification protocol, this phenomenon ischaracteristic of some proteins associated with membranes.

SEQUENCING:

A batch of about 60 μg of polyamidase, prepared by the standard protocolbut starting from 60 g of cells and replacing the Phenylsuperose stepwith a second MonoQ HR 10/10 step, is digested directly with 5 μg of theenzyme LysC in the presence of 0.25% w/v of SDS. The fragments obtainedwere purified on a Vydac C18 column.

The sequence SEQ ID NO: 1 was obtained using conventional nucleotidesequencing techniques.

EXAMPLE 4: ENZYMATIC HYDROLYSIS OF OLIGOMERS FORMED BY MONOMERS A AND BJOINED TOGETHER BY SECONDARY AMIDE LINKAGES, WITH THE AID OF A MIXTUREOF ENZYMES ACCORDING TO THE INVENTION AND PAM I HYDROLASE

Instead of the pure enzymes, the present Example utilizes theirbiological precursors, namely the strain Comamonas acidovorans N 12according to the invention and a strain accommodating the pam I gene(SEQ ID NO: 4 attached) and producing the, enzyme PAM 1 (SEQ ID NO : 3attached), said strain being constructed according to the protocol givenbelow.

The aim of this construction is to obtain the pam I gene (coding for theenzyme-PAM I), which is preceded by the ribosome binding site of thephage λ cll gene and which is expressed from the E. coli tryptophanoperon promoter. To do this, an Ndel restriction site was created at thepam I initiation codon by the PCR technique using, as template, plasmidpXL2297 (FIG. 1) accommodated by the strain I 1495 deposited in the CNCMon Nov. 29, 1994. The 208 bp Ndel-Apal fragment containing the 5′ end ofthe pam I gene was amplified by PCR, care being taken to introduce aHindlll site upstream of the Ndel site by means of the pair ofnucleotide primers (5′-AGCAAGCTTGGAGGCCATATGAATAC GAC-3′) and(5′-CACCGGTGGGCCCCTC-3′). The amplified Hindlll-Apal fragment was clonedinto pUC29 (Benes et al. (1993), Gene 130: 151-152) digested by Hindllland Apal, and the pam I gene was reconstituted by introducing the 867 bpApal fragment of pXL2297 at the Apal site. An Ncol site is thus locatedabout thirty nucleotides downstream of the pam I stop codon. Theadjacent 500 bp Ndel-EcoRl and 600 bp EcoRI-Ncol fragments of thisplasmid were inserted into pXL2158 (patent FR 92-09 882) digested at theNdel site located immediately downstream of the tryptophan promoter.

Thus plasmid pXL2564 (described in FIG. 2) is a derivative of pBR322(Sucliffe (1978), Nucleic Acid Res. 5 : 2721) containing a geneconferring ampicillin resistance and the pam I gene under the control ofthe Ptrp-RBScll expression cassette.

Plasmid pXL2564 was introduced into the E. coli strain TG1, themicroorganisms being selected on ampicillin LB. A clone containingpXL2564 (called strain PAM I) was transferred twice onto agar dishes andcultured at 37° C. in M9 glucose medium containing 100 μg/ml ofampicillin, according to the procedure described in patent FR 2 694 571.

The strain Comamonas acidovorans N 12 was cultivated for 19 hours underthe same conditions as those described in section 2.1 above.

A batch of oligomers was used which consisted of monomers A=adipic acidand B=hexamethylenediamine (HMD) and had a mean DP_(n) of 4.

25.25 g of oligomers with a mean DP_(n) of 8 are resuspended in 300 mlof water. 200 ml of water containing the cells (i.e. 3.1 g in total) areadded. The whole is stirred for 18 hours under a stream of nitrogen.

The solution is centrifuged and the supernatant (400 ml) is recovered. Adry weight of 14 g/l is measured on the supernatant. A fraction is takenfor analysis of the oligomers of adipic acid and HMD.

The results of this analysis are presented in Table 2 below.

TABLE 2 COMPOUND CONTENT DETERMINED (g/l) HMD 13.8 ADIPIC ACID 20 AB 1BAB 3.6 ABA 0.7 ABAB 0.3

The molar yield of adipic acid monomer formed is 66% and the molar yieldof HMD is 58%.

The molar yield of monomers and oligomers with a DP of less than 4,expressed in mol of monomers, is 72%.

4 1 421 PRT Comamonas acidovorans 1 Met Asn Arg Thr Tyr His Arg Arg AspVal Leu Arg Ile Leu Gly Val 1 5 10 15 Gly Thr Ala Leu Gly Gly Ala AlaLeu Leu Gly Ala Cys Gly Gly Ser 20 25 30 Gly Gly Asn Glu Ala Pro Arg GluGln Ile Ala Ser Ser Leu Phe Ser 35 40 45 Thr Thr Pro Glu Asn Arg Ala AlaThr Phe Arg Asn Ala Asp Arg Ile 50 55 60 Val Tyr Ser Arg Thr Ile Lys ArgGly Ala Thr Thr Met Pro Leu Lys 65 70 75 80 Pro His His Val Ser Leu AlaSer Leu Thr Tyr Asp Tyr Ala Gly Lys 85 90 95 Thr Thr Asn Val Asp Asp TyrMet Gln Arg Asn Arg Thr Ala Gly Leu 100 105 110 Leu Ile Leu Lys Gly GlyAla Val Ala Leu Glu Arg Tyr Gly Met Gly 115 120 125 Asn Thr Glu Thr SerArg Trp Thr Ser Trp Ser Val Ala Lys Ser Val 130 135 140 Thr Ser Thr LeuVal Gly Ala Ala Leu Lys Asp Gly His Ile Ala Ser 145 150 155 160 Leu AspAsp Pro Val Thr Arg Tyr Val Thr Ala Leu Lys Gly Ser Ala 165 170 175 TyrGlu Gln Asn Thr Ile Arg Glu Leu Leu Arg Met Thr Ser Gly Val 180 185 190Arg Trp Ile Glu Ala Tyr Ser Glu Thr Gly Asn Ser Asp Ile Ala Arg 195 200205 Leu Arg Glu Ala Tyr Ser Ser Gly Lys Ser Gly Ser Val Met Glu Leu 210215 220 Met Arg Thr Arg Pro Arg Ala Ala Ala Pro Gly Ser Val Phe Asn Tyr225 230 235 240 Ser Thr Gly Glu Ser Tyr Val Leu Gly Ala Val Val Ala AlaAla Thr 245 250 255 Gly Thr Thr Leu Ser Asp Tyr Phe Ser Arg Lys Val TrpAla Pro Phe 260 265 270 Gly Met Glu Ala Asp Gly Tyr Trp Gln Leu Asp SerGlu Gly Gly Leu 275 280 285 Glu Met Gly Gly Ala Asn Phe Ser Ala Thr LeuArg Asp Tyr Gly Arg 290 295 300 Phe Gly Leu Phe Phe Ser Arg Glu Gly ValVal Asn Gly Thr Ala Val 305 310 315 320 Leu Pro Leu Gly Trp Arg Ala LeuAla Ser His Pro Asp Ser Pro Val 325 330 335 Thr Asn Tyr Gly Ala Leu TyrLys Asp Tyr Pro Leu Gly Tyr Gly Tyr 340 345 350 Gln Trp Trp Ala Leu ProGly Lys Asp Thr Thr Ile Pro Ala Gln Asp 355 360 365 Arg Pro Phe Thr AlaGln Gly Ile Tyr Gly Gln Phe Ile Tyr Ile Asp 370 375 380 Pro Lys Glu AspVal Val Ala Val Val Trp Ser Ala Trp Asn Asn Ser 385 390 395 400 Trp ValAsp Ser Ala Glu Phe Glu Thr Phe Ala Leu Leu Ser Lys Ala 405 410 415 ValGlu Met Leu Lys 420 2 1263 DNA Comamonas acidovorans 2 atgaacaggacataccaccg tcgcgatgtg ctgagaattt tgggtgttgg aactgcactt 60 ggaggcgcggcgcttctcgg cgcctgtggc ggcagtggag gcaacgaagc gcctcgggaa 120 caaattgcatcgagcctatt cagcacaact cccgaaaacc gggcggcaac tttccggaat 180 gccgaccgaattgtttactc acgcaccatc aagcgtggcg ccacgaccat gcctctgaag 240 ccgcaccatgtctcgctggc gtccctcaca tatgactatg cggggaaaac cactaacgtg 300 gatgactacatgcagcgcaa tcgcacagct ggattgctta tcttgaaagg cggagcagtc 360 gcgctggagcgctatggcat gggcaacacc gaaacgtccc ggtggacttc atggtcagtc 420 gccaaatctgtcacctccac cttggttggc gcagcgctga aggatgggca cattgccagc 480 ctagacgaccctgtgacgag gtatgtgacg gctttaaaag gcagcgcgta tgaacagaac 540 acaatacgtgagctgctacg gatgacttcc ggcgtacgtt ggattgaagc ctacagcgag 600 acgggcaactccgacattgc ccgactgaga gaggcgtata gttccgggaa aagcggcagc 660 gtgatggagctgatgcgcac acgcccgcgt gcggcagccc ctggcagtgt gtttaactac 720 agcacaggggagagttacgt gctaggcgca gtagttgcag cggccactgg cacaactttg 780 agtgattatttctcccggaa agtatgggca ccgttcggca tggaggccga tggctattgg 840 cagctggattccgaaggagg actggaaatg gggggcgcaa atttcagcgc gaccttgcga 900 gactacgggaggttcggctt gttcttctcg cgcgaaggcg tcgtcaatgg cactgctgtt 960 ctgccgcttgggtggcgggc tcttgctagc catcccgatt cgccagtaac caactacgga 1020 gctctttacaaagactaccc gcttggctat ggataccaat ggtgggcact cccgggcaaa 1080 gatacaacaattccagctca agaccgcccc ttcaccgctc aaggcatcta cggtcagttc 1140 atttacatcgatcccaagga ggatgttgtt gccgtagtgt ggagtgcgtg gaacaactca 1200 tgggtcgacagcgccgagtt tgaaacgttt gcacttctct cgaaggccgt agaaatgttg 1260 aaa 1263 3355 PRT Comamonas acidovorans 3 Met Asn Thr Thr Pro Val His Ala Leu ThrAsp Ile Asp Gly Gly Ile 1 5 10 15 Ala Val Asp Pro Ala Pro Arg Leu AlaGly Pro Pro Val Phe Gly Gly 20 25 30 Pro Gly Asn Asp Ala Phe Asp Leu AlaPro Val Arg Ser Thr Gly Arg 35 40 45 Glu Met Leu Arg Phe Asp Phe Pro GlyVal Ser Ile Gly Ala Ala His 50 55 60 Tyr Glu Glu Gly Pro Thr Gly Ala ThrVal Ile His Ile Pro Ala Gly 65 70 75 80 Ala Arg Thr Ala Val Asp Ala ArgGly Gly Ala Val Gly Leu Ser Gly 85 90 95 Gly Tyr Asp Phe Asn His Ala IleCys Leu Ala Gly Gly Ala Cys Tyr 100 105 110 Gly Leu Glu Ala Gly Ala GlyVal Ser Asp Ala Leu Leu Glu Arg Leu 115 120 125 Glu His Arg Thr Gly PheAla Glu Leu Gln Leu Val Ser Ser Ala Val 130 135 140 Ile Tyr Asp Phe SerAla Arg Ser Thr Ala Val Tyr Pro Asp Lys Ala 145 150 155 160 Leu Gly ArgAla Ala Leu Glu Phe Ala Val Pro Gly Glu Phe Pro Gln 165 170 175 Gly ArgAla Gly Ala Gly Met Ser Ala Ser Ala Gly Lys Val Asp Trp 180 185 190 AspArg Thr Glu Ile Thr Gly Gln Gly Ala Ala Phe Arg Arg Leu Gly 195 200 205Asp Val Arg Ile Leu Ala Val Val Val Pro Asn Pro Val Gly Val Ile 210 215220 Val Asp Arg Ala Gly Thr Val Val Arg Gly Asn Tyr Asp Ala Gln Thr 225230 235 240 Gly Val Arg Arg His Pro Val Phe Asp Tyr Gln Glu Ala Phe AlaGlu 245 250 255 Gln Val Pro Pro Val Thr Glu Ala Gly Asn Thr Thr Ile SerAla Ile 260 265 270 Val Thr Asn Val Arg Met Ser Pro Val Glu Leu Asn GlnPhe Ala Lys 275 280 285 Gln Val His Ser Ser Met His Arg Gly Ile Gln ProPhe His Thr Asp 290 295 300 Met Asp Gly Asp Thr Leu Phe Ala Val Thr ThrAsp Glu Ile Asp Leu 305 310 315 320 Pro Thr Thr Pro Gly Ser Ser Arg GlyArg Leu Ser Val Asn Ala Thr 325 330 335 Ala Leu Gly Ala Ile Ala Ser GluVal Met Trp Asp Ala Val Leu Glu 340 345 350 Ala Gly Lys 355 4 1068 DNAComamonas acidovorans 4 atgaatacga caccggtcca cgcactcacc gacatcgacggcgggatcgc cgtcgatccc 60 gcaccccggc tggccggccc tccggtcttc gggggtccgggcaacgacgc cttcgatctc 120 gcgccggtca ggagcacggg ccgcgagatg ctgcggttcgacttccccgg ggtcagcatc 180 ggcgcggcgc actacgagga ggggcccacc ggtgcgaccgtgatccacat ccccgccggc 240 gcccgcaccg cggtggacgc gcggggcggg gcggtggggctctccggcgg ctacgacttc 300 aaccacgcca tctgcctggc cggcggagcc tgctacgggctcgaggcggg cgccggggtg 360 agcgacgcgc tcctggaacg cctcgagcat cgcaccggcttcgccgagct ccagctggtg 420 tcgtcggcgg tcatctacga cttctcggcg cgctccaccgcggtctaccc cgacaaggcg 480 ctcggccgcg cggcgctcga attcgccgtt cccggtgagttcccgcaggg gcgggcgggc 540 gcgggcatga gcgcgtccgc gggcaaggtg gactgggaccgcaccgagat caccgggcag 600 ggcgcggcgt tccgtcgtct cggcgacgtg cgcatcctcgccgtcgtcgt gccgaacccg 660 gtcggtgtga tcgtggaccg cgcgggcacg gtggtgcgcggcaactacga cgcgcagacc 720 ggggtccggc gccacccggt gttcgactac caggaggcgttcgccgagca ggtcccgccc 780 gtcaccgagg ccggcaacac cacgatcagc gcgatcgtcacgaacgtgcg gatgagcccc 840 gtcgagctga accagttcgc caagcaggtg cacagttcgatgcaccgcgg catccagccg 900 ttccacaccg acatggacgg cgacacgctc ttcgccgtcaccaccgacga gatcgatctg 960 ccgacgaccc cggggtcgtc gcgcgggcgg ctgtcggtgaacgcgaccgc gctcggcgcg 1020 atcgcctccg aggtgatgtg ggacgccgtc ctcgaggccggcaagtag 1068

What is claimed is:
 1. An isolated or substantially purified enzyme with amidase activity, which is a native Comamonas bacterium enzyme with amidase activity or an enzyme expressed by a recombinant bacterium comprising a DNA sequence encoding a native Comamonas bacterium enzyme with amidase activity, said enzyme being active with respect to (poly)amide substrates having the following formula (I):

in which: A and B are monomer units, R¹ and R³ are identical or different divalent radicals representing a substituted or unsubstituted, linear or branched (cyclo)alkylene, an arylene, or an arylalkylene, the aromatic radicals optionally being polycondensates and the number of carbons in the alkylenes being greater than or equal to 4, R² corresponds to identical or different radicals selected from hydrogen and alkyl radicals having from 1 to 6 carbons, X is either X¹ is OH, OM or OR⁴, where M is selected from metals, and R⁴ is a linear or branched alkyl comprising from 1 to 6 carbon atoms, or X² is

where R² and R³ are as defined above and R⁵ and R⁶, which are identical or different, have the same definition as that given above for R², Y is: either Y¹ is hydrogen, or Y² is

where R¹ is as defined above and Z is hydrogen, M¹ defined in the same way as M, or R⁴, wherein if X is X¹, then Y is Y¹ or Y², or, if X is X², then Y is Y², and p is between 1 and 4; and it is capable of converting said substrate (I) to monomers A and monomers B.
 2. The isolated or substantially purified enzyme with amidase activity, according to claim 1, comprising the peptide sequence as shown in SEQ ID NO:
 1. 3. The isolated or substantially purified enzyme according to claim 1, which is capable of hydrolyzing substrate (I) containing at least one carboxyl end group.
 4. The isolated or substantially purified enzyme according to claim 1, wherein: it is active towards a substrate (I) formed by a tetramer of the type ABAB, and it is capable of converting this substrate to a trimer ABA+A with a specific enzymatic activity (U_(s))—expressed in μmol of hydrolyzed ABAB×h⁻¹×mg⁻¹—of greater than or equal to
 1000. 5. An isolated or substantially purified enzyme according to claim 1, wherein: it is active towards a substrate (I) formed by a trimer ABA, and it is capable of converting this trimer to a dimer AB and a monomer A with a specific enzymatic activity (U_(s))—expressed in μmol of hydrolyzed AB/h/mg of enzyme—of greater than or equal to
 1000. 6. An isolated or substantially purified enzyme according to claim 1, wherein: it is active towards a substrate (I) formed by a dimer of the type AB, and it is capable of converting this dimer to two monomers A and B with a specific enzymatic activity (U_(s))—expressed in μmol of hydrolyzed AB/h/mg of enzyme—of greater than or equal to
 1500. 7. An isolated or substantially purified enzyme according to claim 1, wherein it is produced by microorganisms of the Comamonas acidovorans (N 12) type or of the same type as the strain referenced and deposited in the Collection Nationale de Cultures de Micro-organismes under no. I 1522 on Jan. 4, 1995, and/or by their recombinant microorganisms.
 8. An isolated or substantially purified enzyme with amidase activity, which is a native Comamonas bacterium enzyme with amidase activity or an enzyme expressed by a recombinant bacterium comprising a DNA sequence encoding a native Comamonas bacterium enzyme with amidase activity, said enzyme being active with respect to (poly)amide substrates having the following formula (II):

in which: a, b and c are monomer units, R² corresponds to identical or different radicals selected from hydrogen and alkyl radicals having from 1 to 6 carbons, and R³ is identical or different divalent radicals representing a substituted or unsubstituted, linear or branched (cyclo)alkylene, an arylene, or an arylalkylene, the aromatic radicals optionally being polycondensates and the number of carbons in the alkylenes being greater than or equal to 4, U is OH, OM or OR⁴, where M is selected from metals, and R⁴ is a linear or branched alkyl comprising from 1 to 6 carbons, and V is hydrogen, and q is between 1 and
 8. 9. The isolated or substantially purified enzyme according to claim 8, which is capable of hydrolyzing substrate (II) containing at least one carboxyl end group.
 10. An isolated DNA fragment containing a DNA sequence coding for an enzyme with amidase activity, which is selected from the sequences of: the DNA sequence as represented by SEQ ID NO: 2 and coding for at least one enzyme with amidase activity, and an analog of this sequence which results from the degeneracy of the genetic code.
 11. An isolated or substantially purified enzyme produced by the expression of the DNA sequence according to claim
 10. 12. A microorganism comprising at least one expression cassette comprising the DNA sequence according to claim 10 and optionally, upstream thereof, at least one promoter sequence and at least one ribosome binding site.
 13. A process for the hydrolysis of (poly)amides, comprising conducting enzymatic hydrolysis on substrates comprising (poly)amides of the following formula (I):

in which: A and B are monomer units, R¹ and R³ are identical or different divalent radicals representing a substituted or unsubstituted, linear or branched (cyclo)alkylene, an arylene, or an arylalkylene, the aromatic radicals optionally being polycondensates and the number of carbons in the alkylenes being greater than or equal to 4, R² corresponds to identical or different radicals selected from hydrogen and/or alkyl radicals advantageously having from 1 to 6 carbons, X is: either X¹ is OH, OM or OR⁴, where M is selected from metals, and R⁴ is a linear or branched alkyl comprising from 1 to 6 carbon atoms, or X² is

where R² and R³ are as defined above and R⁵ and R⁶, which are identical or different, have the same definition as that given above for R², Y is: either Y¹ is hydrogen, or Y² is

where R¹ is as defined above and Z is either hydrogen, M¹ defined in the same way as M, above, or R⁴ as defined above, wherein if X is X¹, then Y is Y¹ or Y², or, if X is X², then Y is Y², and p is between 1 and 4; and obtaining monomers A and B.
 14. The process of claim 1, wherein said amidase enzyme is a native Comamonas bacterium enzyme with amidase activity or an enzyme expressed by a recombinant bacterium comprising a DNA sequence encoding a native Comamonas bacterium enzyme with amidase activity, said enzyme being active with respect to (poly)amide substrates having formula (I).
 15. The process of claim 1, wherein said amidase enzyme has the amino acid sequence encoded by the nucleic acid of SEQ ID NO:2.
 16. A process for hydrolysis of substrates comprising oligomers whose degree of polymerization (DP_(n)) is less than 40, which oligomers are derived from polyamides resulting from polycondensation of between diacid monomers (A) and diamine monomers said process produces one or more of monomers A, monomers B, and oligomers with a DP_(n) less than or equal to 3, and, wherein said process utilizes at least one enzyme according to claim 1, as well as at least one other enzyme, said at least one other enzyme selected from the group consisting of: the enzyme E3 produced either in its wild-type form or biological precursor thereof, or as a product of recombinant expression or biological precursor thereof of the nyl-c gene of Flavobacterium so. K 172, and, the enzyme PAM I as defined by the amino acid sequence of SEQ ID NO:3 or the enzyme produced under the control of the microorganism referenced and deposited in the Collection Nationale de Cultures de Micro-olganismes—Institut Pasteur PARIS—under No. I 1495 on Nov. 29, 1994, either in its wild-type form or biological precursor, or as a product of recombinant expression or biological precursor.
 17. A process for the hydrolysis of substrates at least partly formed by substrate (I) as defined, wherein said process comprises using at least one enzyme according to claim
 1. 18. A process for the hydrolysis of substrates at least partly formed by substrate (II) as defined in claim 4, wherein said process comprises using an enzyme according to claim
 4. 19. The process of claim 18, wherein substrate (II) is partially hydrolyzed.
 20. The process of claim 18, wherein the hydrolysis of substrate (II) includes at least one other enzyme. 